ISSN 1646-6551

Clinical Journal of

January 2014 Volume IV

The Clinical Journal of Mycology is dedicated to the dissemination of information on the clinical use of nutrition to health care practitioners.

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­— Page 2 Comparative Differences in β-1,3-1,6 Glucan content between Ganoderma lucidum (Reishi) (Biomass vs Extracted) in the Presence of Proteolytic Enzymes. Prof. Amin Karmali – Chemical Engineering and Biotechnology Research Center and Department of Chemical Engineering of Instituto Superior de Engenharia de Lisboa (ISEL), Lisbon Polytechnic Institute ,Portugal.

— Page 7 Coriolus-MRL supplementation in patients infected with low-risk and high-risk HPV subtypes-Bulgarian experience.

Prof. Todor Chernev – University Hospital of Obstetrics and Gynecology, Sofia, Bulgaria

— Page 8 Coriolus versicolor – Innovation in Prevention of Oncogynecological Diseases, especially HPV.

Dr. J. Bogdanova – Institute of Botany, Bulgarian Academy of Sciences, Sofia, Bulgaria

— Page 10 Link between Herpes Simplex Virus and Alzheimer´s Disease: Potential Role of Mushroom Nutrition Supplementation in Prevention. Prof. Tito Fernandes – Faculty of Health Sciences, Lurio University, Nampula, Mozambique. Prof. Vittorio Calabrese – Faculty of Medicine, University of Catania, Catania, Italy

£7.00 $14.00 €10.50 Comparative Differences in ß-1,3-1,6 Glucan content between Ganoderma lucidum (Reishi) mushrooms (Biomass vs Extracted) in the Presence of Proteolytic Enzymes

Professor Amin Karmali Email: [email protected] Institution: Centro de Investigação de Engenharia Quimica e Biotechnologia-Lisbon Polytechnic Institute- Portugal.

INTRODUCTION Some mushrooms have been known to exhibit several form). The Reishi biomass form demonstrated a greater medicinal properties for thousands of years in Japanese overall enzyme activity level over the extracted form of and other Asian cultures. The Reishi mushroom, also known Reishi. as Ganoderma lucidum, is well characterized (in Traditional It has been known for over a century that some enzymes Chinese Medicine) for the prevention and treatment of several can be used in the prevention and even treatment of several disease states such as cancer, allergies and asthma. clinical conditions. These enzymes are divided into the In the West, Reishi is sold in an extracted form (extracted following activities:: specifically for β-glucan content) or in a biomass form Enzymes that prevent oxidative stress: ( and primordial (young fruit body)). The biomass Superoxide dismutase form contains several substances of clinical interest such as enzymes, secondary metabolites and β-glucans. The specific Enzymes that prevent cellular growth: quantification ofβ -glucans in mushrooms (extracted or biomass Protease (1) forms) with anti-tumour activity is of great clinical importance Glucoamylase There are several types of β-glucans in mushroom species such Enzymes that promote detoxification: as β-1,3 glucans and β-1,3-1,6 glucans(2). As far as anti-tumour immune enhancing and modulating activities, these three Cytochrome P-450 activities are attributed to β-1,3-1,6 glucans which exhibit a Peroxidase (3,4) triple helix as their tertiary structure . 2 oxidase Methods Reishi Myco exhibits low levels of secondary metabolites Two samples (1g) of Reishi MRL and Reishi Myco were compared with the Reishi-MRL product. compared to detect and to quantify enzymes, β-glucans and secondary metabolites. These two samples are different since Conclusions the former is a biomass that contains mycelia and primordia The differences in β-1,3-1,6 glucan content between both whereas the latter is a concentrated extract (20x) of fruiting samples may be due to differences in biological material bodies. in these samples since one contains mycelia and primordia The β-1,3-1,6 glucan content was determined by a colorimetric whereas the other consists of concentrated (20x) extract method recently established in Germany (2). The enzyme and of fruiting bodies. In fact, immunonutrients in mycelia and secondary metabolite contents were determined by methods primordia (young fruiting bodies) in MRL product are more commonly used. resistant to proteolytic enzymes (i.e simulation of digestive tract) since it is in a biomass form and not on cell extract. In order to assess the impact of digestive enzymes on the Therefore, the concentrated extract of the fruiting bodies constituents of each sample, both samples were compared is more exposed and available to the action of proteolytic in vitro; a) in the absence of proteolyic enzymes, b) in the enzymes (i.e simulation of digestive tract) since there are no presence of pepsin and c) in the presence of trypsin. physio-chemical barriers to prevent such exposure. In addition to the importance of β-1,3-1,6 glucan Discussion content, one should assess the advantages of enzyme The data obtained reveal that, in the absence of proteolytic supplementation afforded by the biomass form of mushroom enzymes, both forms of Reishi contain significant levels of nutrition. β 1,3-1,6 glucans with anti-tumour activity with Reishi Myco exhibiting higher values (hot water fraction and NaOH fraction).However, in the presence of pepsin and trypsin, Reishi-MRL (biomass form) exhibited higher β1,3-1,6 glucan values than Reishi Myco (extracted form) (See Table I and Fig. 7-11). When comparing the enzyme values, in the absence of, and presence of, proteolytic enzymes, between the two forms of Reishi, (See Table II and Figures 1-6), there was an absence of important immune-enhancing enzyme activity, such as peroxidase, glucoamylase and protease activities in Reishi Myco (extracted form) when compared to Reishi-MRL (biomass continued…

2 Professor Amin Karmali

Fig. 1- Peroxidase Fig. 4 - Glucose 2-Oxidase

Fig. 2- Glucoamylase Fig. 5 - SOD

Fig. 3 - Protease Fig. 6 - Cytochrome 450

3 Comparative Differences in ß-1,3-1,6 Glucan content between Ganoderma lucidum (Reishi) mushrooms (Biomass vs Extracted) in the Presence of Proteolytic Enzymes

Professor Amin Karmali [email protected]

Fig. 7 - Water Soluble Fraction Fig. 10- KOH Fraction

Fig. 8 - Hot Water Fraction Fig. 11 - HCL Fraction

Fig. 9 - NaoH Fraction

Clinical Journal of Mycology is published by Aneid Press, a division of Aneid Lda Editorial Advisors: Tito Fernandes DVM,MSc,PhD,DSc,Dr HC mult, Dip ECVCN, AAVN. (Portugal) Design & Production: Allan Parker Publishing Director: William Ahern ISSN 1646-6551

4 continued…

Table I: Comparision of Impact of Proteolytic Enzymes on Beta 1,3-1,6 Glucan Activity between Reishi-MRL (biomass) and Reishi Myco (extract) (one gram of product in powder form)

I. IN ABSENCE OF PROTEOLTIC ENZYMES (per gram of product)

In absence of Proteolytic Enzymes Reish A MRL Reshi B Myco

2.1 Water soluble fraction 24.0µg 117.0µg

2.2 Hot water fraction 29.0µg 750.0µg

2.3 NaOH fraction 976.0µg 2193.0µg

2.4 KOH fraction 2213.0µg 246.0µg

2.5 HCl fraction 642.0µg 378.0µg

ii In the presence of Pepsin (per gram of product)

Reish A Reshi B

2.1 Water soluble fraction 21.0µg 11.5µg

2.2 Hot water fraction 22.0µg 5.0µg

2.3 NaOH fraction 956.0µg 21.9µg

2.4 KOH fraction 2103.0µg 26.0µg

2.5 HCl fraction 632.0mg 33.0mg

iii In the presence of TRYPSIN (per gram of product)

Reish A Reshi B

2.1 Water soluble fraction 21.0µg 11.2µg

2.2 Hot water fraction 25.0µg 7.1µg

2.3 NaOH fraction 962.0µg 21.93µg

2.4 KOH fraction 2113.0µg 24.7µg

2.5 HCl fraction 630.0µg 32.0µg

5 Comparative Differences in ß-1,3-1,6 Glucan content between Ganoderma lucidum (Reishi) mushrooms (Biomass vs Extracted) in the Presence of Proteolytic Enzymes

Professor Amin Karmali [email protected]

Table II: Comparison of Impact of Proteolytic Enzymes on Beta 1,3-1,6 Glucan , Enzyme and Secondary Metabolite Activity between Reishi-MRL (biomass) and Reishi Myco (extract) in one gram of powder.

Enzymes, polysacharides and secondary In absence of Proteolytic Enzymes In presence of Pepsin In presence of Trypsin metabolites per gram of product

Reish A MRL Reshi B Myco Reish A MRL Reshi B Myco Reish A MRL Reshi B Myco

1 Protein content 44.8mg 40.5 mg 35.9 mg 30.5mg 37.1mg 33.2mg

2 b-1,3-1,6- glucans with anti-tumour activity

2.1 Water soluble fraction 24.0mg 117.0mg 21.0mg 11.5mg 21.0mg 11.2mg

2.2 Hot water fraction 29.0mg 750.0mg 22.0mg 5.0mg 25.0mg 7.1mg

2,3 NaOH fraction 976.0mg 2193.0mg 956.0mg 21.9mg 962.0mg 21.93mg

2.4 KOH fraction 2213.0mg 246.0mg 2103.0 mg 26.0mg 2113.0mg 24.7mg

2.5 HCl fraction 642.0mg 378.0mg 632.0mg 33.0mg 630.0mg 32.0mg

3 Peroxidase activity 20.9mU 0.0mU 18.3mU 0.0mU 18.7mU 0.0mU

4 Glucoamylase/Beta-glucanasase activity 5.3 U 0.0 U 4.8 U 0.0mU 4.9 U 0.0mU

5 Protease activity 9.1mU 1.1mU 8.4mU 1.0mU 8.5mU 0.8mU

6 Glucose 2-oxdase activity 14.3mU 10.1mU 12.1mU 7.2mU 13.2mU 8.5mU

7 Superoxide dismutase (SOD) activity 98.4mU 99.8mU 82.3mU 71.7mU 87.5mU 75.9mU

8 Cytochrome “P-450” 1.4 nmoles 1.5nmoles 1.3nmoles 1.1nmoles 1.2nmoles 1.0nmoles

9 Cytochrome P 450 reductase 15.5mU 12.5mU 12.6mU 8.7mU 13.3mU 6.2mU

10 Secondary metabolites (Thrombin inhibitors) 4.9% 1.1% 4.6% 1.0% 4.7% 1.0%

References 1. Production, purification and characterization of Note: One enzyme unit (U) is defined as the amount of enzyme required to convert from ostreatus with antitumour activity one micromole of substrate to product per minute under certain experimental con- Silva S, Martins S, Karmali A, Rosa E.J Sci. Food Agric. 2012;92(9): ditions. One milli-enzyme unit (mU) is defined as the amount of enzyme required to 1826-32. convert one nanomole of substrate to product per minute under certain experi- 2. A new colorimetric method to quantify β-1,3-1,6-glucans in mental conditions comparison with total β-1,3-glucans and a method to quantify chitin in *Centro de investigação de Engenharia Química e Biotecnologiaia - ISEL edible mushrooms Helga Mölleken, Jörg Nitschke, Hendrik Modick, Tim Malolepszy, Hans-josef Altenbach; Food chemistry 127 (2011), 791-796. 3. The structure and conformation of a water-insoluble (1->3)-, (1->6)-beta-d-glucan from the fruiting bodies of Pleurotus florida. Rout D, Mondal S, Chakraborty I, Islam SS. Carbohydr Res. 2008; 343(5):982-7. 4. Glucans from the alkaline extract of an , Pleurotus florida, cv Assam Florida: isolation, purification, and characterization. Ojha AK, Chandra K, Ghosh K, Islam SS. Carbohydr Res. 2010 ; 345(15) :2157-63

6 Coriolus-MRL supplementation in patients infected with low-risk and high-risk HPV subtypes - Bulgarian experience

Prof. Todor Chernev, [email protected] University Hospital of Obstetrics and Gynaecology – Sofia, Bulgaria

Introduction: Results: The results showed that 64 (88%) out of 73 patients on Coriolus-MRL is a food supplement which contains biomass of the conservative treatment and 25 (93%) out of 27 patients on combined Coriolus versicolor. Recent studies showed a positive effect of treatment were HPV-negative. 4 this non-specific immunomodulator in HPV patients . The specialists in All PAP group IIIa and IIIb patients reverted to PAP group I and/or II Bulgaria have a five-year experience with the use of Coriolus-MRL and (Table 1). the results show high effectiveness in HPV carrying patients1,2. At the end of the study only 11 patients were still positive for 1 or more HPV subtypes (Table 2). HPV subtype 16 was found to be most treatment resistant 2.

Study 2 (2010-2012) Materials and methods: 200 patients; Period of treatment: 6 months; Treatment: Coriolus-MRL 2 x 3 tablets. Results: 95% of the patients reverted to HPV-negative status. HPV subtype 16 was again most resistant. HPV-positive patients without Study 1 (2009-2010) histological changes reverted to HPV-negative status in 3 months. In patients with mild dysplasia (CIN I) Coriolus-MRL supplementation can Materials and methods: help the body clear the infection. In cases with severe dysplasia (CIN II 100 women aged 16-50, infected with low-risk and high-risk HPV and CIN III) Coriolus-MRL supplementation can strengthen the immune subtypes; Period of treatment: 6 months, system and reduce the risk of relapse. Conservative treatment: Coriolus-MRL 2 x 3 tablets; Coriolus-MRL have been taken also by the women’s partners in about Combined treatment: Surgical intervention + Coriolus-MRL 2x3 tablets; 70% of the couples. 90% of these couples reverted to HPV-negative status after 6 months1. 1st month 6th month Negative Positive Low-risk Table 1. Results of a 6-month study: number of the patients and the PAP smears HPV subtypes 4 4 – High-risk HPV subtypes 96 85 11 in the respective groups. Conclusions: Coriolus-MRL has no adverse reactions and drug interactions with 1st month 6th month the main drugs used for the treatment and it could be taken during PAP I PAP II PAP III a pregnancy. It is an addition to the methods for prevention and treatment (both combined and conservative) of HPV infection 47 47 – PAP I PAP II References 29 PAP III a 9 20 – 1.The importance of the immunity in the treatment of HPV infections. Tcholakova A. MEDINFO 2012 (2), 28-29. e-mail: [email protected] 9 PAP III b 3 6 – 2. Assessment of the effect on patients infected with low and high-risk types 15 PAP III d 6 6 3 of HPV. Borisov S. 2012. Coriolus-MRL Clinical Journal of Mycology, Vol.3, Ed.2, 2-3. e-mail: [email protected] 100 3. Coriolus versicolor: a medicinal mushroom with promising immunotherapeutic values Chu K., Ho S., Chow A. (2002).. Journal of Clinical Pharmacology, 42: 976-984. Table 2. Results of a 6-month study: patients who were found to carry high-risk or low-risk HPV subtypes. 4. Evaluation of Coriolus versicolor Supplementation in HPV Patients. Couto S. (2007). Clinical Journal of Mycology, Vol. 2, Ed. 1:2-5.

1st month 6th month e.mail: [email protected]

Negative Positive

Low-risk HPV subtypes 4 4 –

High-risk HPV subtypes 96 96 11

7 Coriolus versicolor – Innovation in Prevention of Oncogynecological Diseases, especially HPV.

Yuliyana Bogdanova, PhD Email: [email protected] Institute: Institute of Botany, Bulgarian Academy of Sciences, 2 Chernorizec Hrabar Str., app.11, 1164 Sofia, Bulgaria Tel/Fax +359 2 963 1441

Abstract infected with HPV within 4 years after they start their sexual life, and Coriolus-MRL is a nutrient adjuvant, which contains biomass of the that 90 % of all cases of cervical cancer are caused by HPV. Thirteen out fungus Coriolus versicolor and is studied to reverse early stages of of 100 subtypes of HPV are considered high-risk for the development cervical cancer and to reduce risk factors of reoccurring HPV virus. of cervical cancer. Studies by many authors point out a relation between 7 (PMID:19449722 / Akush Ginekol (Sofiia). 2008; 47 Suppl 3:51-3. HPV and prostate cancer ; some esophagus cancer cases may be also [PubMed – indexed for MEDLINE [Original Article in Bulgarian; the resulting from same virus both in man and women, possibly through following an English Translation approved by author.] oral sex. The anti-HPV vaccines introduced in the 1990s are effective against 2 to 4 of the high-risk subtypes HPV and are effective only in HPV-uninfected Summary Coriolus-MRL is a nutrient adjuvant, which contains biomass of the women. Surgical treatment of HPV lesions is not possible in the early fungus Coriolus versicolor with immune-modulating components stages and in many cases there is a risk of recurrence of the process. – β-glucans and proteoglucans. Research has been carried out in Coriolus versicolor is a fungus whose biomass acts as a non-specific recent years, establishing the effects of the mushroom biomass in the immunomodulator and is a suitable adjuvant in cancer patients for the prevention of HPV patients from developing cervical cancer as well as strengthening of the immune system, especially after chemotherapy the prevention against other cancer-causing viruses. and radiotherapy (1). Immuno-modulating nutrition is a new concept, representing the This medicinal mushroom is widely distributed in the woodlands potential for modulating the activities of the immune system through of North America, Asia and Europe. It is parasitic on deciduous and intake of specific nutrients and strengthening the capability of the coniferous tree species or saprophytic on tree stumps. It consists of organism for self-defense and prevention. mycelium (made up of hyphae) and fruit bodies, where for Fungotherapy (treatment with medicinal mushrooms) is an important reproduction are formed. part of immune-modulating nutrition. The biological activity of the The immunostimulating effect of Coriolus versicolor is due to the fungi is connected with improvement in cardiac activity, strengthening β-glucans and proteoglucans it contains. β-glucans are polysaccharides, of the immune system, alleviation of allergic symptoms, normalization consisting only of glucose residues, and proteoglucans are proteins of sugar blood levels and detoxification of the organism. One of the connected with chains. They have the selective ability most important effects is the prevention from cancer-inducing viruses. to cause apoptosis (programmed cell death) in cancer cells without Such viruses are the Human Herpes virus type 8 (HHV-8), Human damaging the healthy ones (4). papillomavirus (HPV), Epstein-Barr virus (EBV) and hepatitis B virus (HBV)(3). The relation between HPV and the development of cervical cancer Fig.1 Selective ability of the β-glucans to cause apoptosis in cancer has been proved. It has been established that 80% of all women get cells

8 Coriolus versicolor – Innovation in Prevention of Oncogynecological Diseases, especially HPV.

Yuliyana Bogdanova PhD

Back in the 1970s two proteoglucans, typical of this fungus species decreases in viral loads was established. On the basis of these results were isolated – polysaccharide K (PSK) and polysaccharopeptide and previous studies on the influence of folic acid in HPV control, Dr. (PSP). A number of studies prove the activity of these compounds Monro recommends a scheme of intake of Coriolus-MRL and folic acid in stimulating the effect of natural killer cells and the increase of the for 8 weeks with the aim of preventing HPV patients from developing number of T-lymphocytes. cervical cancer and reversing the process of dysplasia. During the The activity of enzymes preventing from oxidizing stress, inhibiting treatment period patients are given 3g Coriolus-MRL daily as well as cellular reproduction and contributing for the detoxification of the 300mg folic acid daily for 1 week and 10mg folic acid daily for 7 weeks. organism was established. Immunostimulating effect is strengthened Control of microbiological and immune markers is recommended in 5 by the content of some secondary metabolites (lectines, terpenoids, order to evaluate the synergistic effect of Coriolus-MRL and folic acid . chelates). Dr. L. J. Standish from the School of Naturopathic Medicine at Bastyr A specially created sort (CV-OH1), a result of 15 years of work University has received funding from the Cancer Treatment Research of scientists from Mycology Research Laboratories and Gourmet Foundation (CTRF) to perform a placebo-controlled clinical trial looking Mushrooms Inc., California, USA is used for the industrial cultivation of at the effects of Coriolus versicolor supplementation on the immune Coriolus versicolor. The mycelium of this sort has very high bioactivity system, quality of life and fatigue of women with breast cancer after and vitality and is genetically identical with the initial parent forms. The completing radiation therapy. cultivation of the fungus and the whole process of tablet production Coriolus-MRL is appropriate as a dietary supplement for patients ensure preservation of β glucans, proteoglucans and enzymes as major with immune deficit after illness and after surgical intervention, for immune-modulating factors. The required microbiological control prevention of cancer-causing viruses and as adjuvant therapy in patients avoids the presence of impurities and accumulation of pesticides and with cervical cancer, breast cancer and prostate cancer. heavy metals in the biomass. The nutrient adjuvant Coriolus-MRL is not an extract; it contains mycelium and young fruit bodies of the fungus. References: In this way the synergistic effect of all biologically active compounds in 1. Chu K., Ho S., Chow A. (2002). Coriolus versicolor: a medicinal mushroom the biomass is ensured. with promising immunotherapeutic values. Journal of Clinical Pharmacology, Over 350 scientific and clinical studies have been published since 42: 976-984. 1971, when polysaccharide K was discovered. The activity of Coriolus versicolor in various diseases, especially oncological diseases was 2. Couto S. (2007). Evaluation of Coriolus versicolor Supplementation in HPV shown. Recent studies have established its positive effect in HPV Patients. Clinical Journal of Mycology, Vol. 2, Ed. 1:2-5. patients. 3. French A. (2007). Coriolus versicolor Supplementation for Recurrent Herpes Dr. Silva Couto from the Institute of Oncology- Coimbra, Portugal carried Simplex. Clinical Journal of Mycology, Vol. 2, Ed. 1: 11-12. out clinical trial on the effect of Coriolus versicolor in HPV patients with 4. Jimenes Medina E., Berruguilla E., Romero I., Algarra I., Collado A., Garrido cervical lesions2. 39 patients with low-grade squamous intraepithelial F., Garcia-Lora A. (2008). The immunomodulator PSK induces in vitro cytotoxic lesions (LSIL), 22 of them with high-risk subtype HPV, were included. activity in tumour cell lines via arrest of cell cycle and induction of apoptosis. Two groups were formed: control group, without any treatment, and BMC Cancer. 24: 8:78. a group with an intake of 6 tablets (3g) Coriolus-MRL per day in the 5. Monro J. (2005). Cytokine Th1 to Th2 Shift can be Reversed by Coriolus. course of 1 year. All patients were submitted to colposcopy, biopsy and Prospective Trial for HPV Control with Coriolus. Clinical Journal of Mycology, HPV tipification in the beginning and at the end of the study. Results Vol. 1, Ed. 10: 4. showed normal cervical cytology in 13 (72.5%) of the 18 women taking 6. Monro J. (2005). Use of Coriolus versicolor in Chronic Fatigue Syndrome. Coriolus-MRL. In the control group the percentage was 47.5%. Of Clinical Journal of Mycology, Vol. 1, Ed. 10: 3. the 10 high-risk patients taking Coriolus-MRL, 9 moved from HPV(+) to HPV(-) status. Only one of the 12 women in the control group was 7. Serth J, Panitz F, Paeslack U, Kuczyk MA, Jonas U. 1999 Increased levels of HPV(-) at the end of the study. This researcher has reported regression human papillomavirus type 16 DNA in a subset of prostate cancers. of dysplasia and reversion from HPV(+) to HPV(-) status in high-risk Cancer Res, 59(4):823-5. patients with LSIL. In patients with high-grade squamous intraepithelial lesions (HSIL), who were still high-risk HPV(+) after conization, Dr Couto Additional Bulgarian Pub Med References to Coriolus-MRL: also reports a positive effect. Akush Ginekol (Sofiia). 2009; 48 Suppl 2:31.[Coriolus-MRL-a new addition to the Dr. Jean Monro – Medical Director in Breakspear Hospital, UK applied arsenal for complex treatment of oncogynecological diseases]. Coriolus-MRL in 36 patients with Chronic Fatigue Syndrome and immune [Article in Bulgarian] Borisov S. PMID: 20380095 [PubMed - indexed for MED- deficiency manifested by irregular differentiation of T-lymphocytes, low LINE] level of natural killer cells, active viruses and high titres of antibiodies, IgG or IgM to a range of viruses6. Patients took Coriolus-MRL – 6 tablets of 500mg for 15 days, and 3 tablets of 500mg for 45 days. At the end of the study a doubling of the levels of natural killer cells and

9 Link between Herpes Simplex Virus and Alzheimer´s Disease Potential Role of Mushroom Nutrition Supplementation in Prevention

Professor Tito Fernandes, [email protected] Scientific Director, Lúrio University, Nampula, Mozambique, Tel: +25826214040; +258829565760

Herpes exists in two common forms. The majority of the population The researchers demonstrated that aspirin–triggered Lipoxin A4 (LXA4) acquires Herpes Simplex Virus (HSV-1), which causes cold sores, during (15 μg/kg) s c, twice a day, reduced NF-kB activation and levels of pro- childhood from non-sexual contact. Herpes Simplex Virus 2 (HSV-2), inflammatory cytokines and chemokines, as well as increased levels of also known as genital herpes, is transmitted by sexual contact. anti-inflammatory IL-10 and transforming growth factor B (beta). Such In 2000, researchers led by Dr. Frank M. LaFerla at the department changes in the cerebral milieu resulted in recruitments of microglia of Neurobiology and Behaviour at the University of California Irvine, in an alternative phenotype as characterized by the up-regulation of Irvine, USA, demonstrated that a synthetic protein that resembles the YM1 and arginase-1 and the down-regulation of inducible nitric oxide (9) Herpes Simplex Virus (HSV-1) mimics the structure and function of a synthase expression. In effect the researchers contend that activating (10) protein called β-amyloid, a toxic agent that accumulates in the brains LXA4 signaling may represent a novel therapeutic approach for AD . of Alzheimer patients.(1) Given the potential gastrointestinal discomfort associated with aspirin Genetic sequencing revealed that two-thirds of the viral protein is intake, is there another manner to achieve to trigger LXA4 as well as provide both anti-viral protection and anti-oxidant protection in the identical to the β-amyloid protein. The researchers showed that, form of SOD (Super-oxide dismutase) supplementation? like β-amyloid, it could destroy brain neurons, a key feature in the development of Alzheimer´s. Moreover, in laboratory experiments, the viral protein formed abnormal twisted fibres (neurofibrillary tangles, Why Mushroom Nutrition? called ‘tau’) like those found in the brains of Alzheimer´s patients-the In the past ten years, the clinical development of mushroom nutrition definitive hallmark of the disease.(2) has determined that Coriolus versicolor (biomass) has viral protective According to Dr. LaFerla “Most people are exposed to HSV-1, but do properties, while erinaceus (biomass) is extremely high in not develop Alzheimer´s. However, recent studies show that people SOD content. genetically predisposed to Alzheimer´s are more likely to develop the Given this information, there are four reasons to consider the use of disease if they are exposed to herpes”.(3) mushroom nutrition in the preventative use for patients with Alzheimer´s In support of Dr. LaFerla´s hypothesis, in December 2008, Professor disease if HSV-1 induced: Ruth Itzhaki and her team in the United Kingdom, at the Manchester Coriolus versicolor has a clinically verified use in the reduction of viruses University´s Faculty of Life Sciences published in the Journal of Pathology (EBV, CMV and HHV-6) related to the Chronic Fatigue Syndrome that the HSV-1 DNA is located very specifically in amyloid plaques: 90% condition(11,12) . of plaques in Alzheimer´s disease sufferers´ brains contain HSV-1 DNA, Coriolus versiclor has been used to increase the regression rate of LSIL (4) and most of the viral DNA is located within amyloid plaques . lesions in HPV patients and to reduce the viral load in HPV patients. (13) The team had previously shown that HSV-1 infection of nerve-type cells Hericium erinaceus has an extremely high SOD content, which in the induces deposition of the main component, β-amyloid, of amyloid presence of proteolytic enzymes per 500 mg tablet has a SOD content plaques. Together these findings strongly implicate HSV-1 as a major of 19.430x103 U(14). This high SOD content is important given that with factor in the formation of amyloid deposits and plaques, abnormalities herpes simplex virus infection, apoE4 intensifies virus latency and is thought by many in the field to be major contributors of Alzheimer´s associated with the increased oxidative damage of the central nervous disease. system, and there is some evidence that herpes simplex virus infection The University of Manchester´s data strongly suggest that HSV-1 has in combination with the apoE4 genotype may be associated with a major role in Alzheimer´s disease and point to the usage of antiviral increased risk of Alzheimer´s disease (AD)(15). agents for treating the disease, in fact in preliminary experiments they For those patients not able to both tolerate the side effects of anti- have shown that acyclovir reduces the amyloid disposition and also HSV1 virals and/or afford intravenous immunoglubin IG therapy, having reduces certain other features of the disease which they have found an alternative. are caused by HSV-1 infection. Further research has been conducted on The following is a proposed two part study to determine if biomass the potential use of acyclovir, penciclovir and foscarnet as therapeutic form of both Coriolus versicolor and Hericium erinaceus are able to agents for the treatment of Alzheimer`s disease (4)(5)(6). trigger LXA4 stimulation of microglia and indirectly reduces Alzheimer In addition to the use of anti-HSV1 antiviral agents to disrupt the HSV1 disease symptoms in early stage patients. Working with the University virus, researchers at the Medical University of South Carolina have of Catania, the protocol structure is the following: suggested the use of immunoglobulin (IG) GM genes based on their Protocol to determine if Coriolus versicolor and Hericium erinaceus putative role as the modulators of host immune response. In some stimulates Lipoxin A4 (LXA4) activation in peripheral blood and in the studies, the intravenous use of immunoglobulin acts synergistically with CNS of rats treated with an equivalent human dose of 3 g per day MRLs the antiviral, acyclovir.(7)(8) given, orally, separately or in combination, morning and evening, for 1 More recently, in May of 2013, Dr. LaFerla and his associates at the and, respectively, 3 months. At the end of experimental period animals Institute of Memory Impairments and Neurological Disorders at will be sacrificed and the activity of LXA4 will be determined in serum, the University of California, Irvine, Irvine California published in the lymphocytes and in different brain regions (cortex, striatum, substantia American Journal of Pathology, that there could be another method to nigra, hippocampus and cerebellum) and compared with LXA4 of reduce Alzheimer´s disease (AD). untreated animals, as control.

10 Link between Herpes Simplex Virus and Alzheimer´s Disease Potential Role of Mushroom Nutrition Supplementation in Prevention

Professor Tito Fernandes, Professor Vittorio Calabrese (right)

Professor Vittorio Calabrese, [email protected] University of Catania, Catania, Italy, Tel:+39 3288310716

A. Patient Selection: 20 control subjects and 20 patients with early stage AD diagnosis will 1. Test for Lyme disease and or associated conditions with Lyme be treated with MRLs (Coriolus versicolor and Hericium erinaceus) for disease.* a period of 180 days (See section III). For each mushroom (Coriolus versicolor and Hericium erinaceus) the supplementation schedule 2. Patients with genetic precondition (based on family history) to should be six tablets (500 mg) per day or 3 g per day (3 tablets in Alzheimer’s. mornings before breakfast and 3 tablets in evening before dinner). 3. Patients with high HSV-1 viral load. (Need to define). Supplementation period: 180 days. After the treatment period blood 4. Cognitive testing to determine a baseline (see point 5). samples will be taken and LXA4 activity will be determined in serum and 5. Determine number of “senior moments” per week. limphocytes together with heme oxygenase-1 expression as marker of anti-inflammatory and antioxidant potential. 6. Supplement for six months and retest against HSV-1 viral load, cognitive baseline test and number of “senior moments” per week. Upon determination of the potential for mushroom nutrition to trigger LXA4 activity, the next step will be to clinically test the hypothesis in early stage Alzheimer´s patients. B. Supplementation Schedule: Protocol Outline for use of Mushroom Nutrition in Early Stage Alzheimer For each mushroom (Coriolus versicolor and Hericium erinaceus) the Patients. supplementation schedule should be six tablets (500 mg) per day or 3 g per day (3 tablets in mornings before breakfast and 3 tablets in evening before dinner). Supplementation period: 180 days. Table I: Proposed Human Trial Protocol and Corresponding Cost per Day for Use of Mushroom Nutrition in AD. C. Success Criteria after 180 days: Testing for:

Coriolus versicolor Hericium erinaaecus 500mg tablets 500mg tablet 6 tablets/day = 3g 6 tablets/day = 3g

Month 1 180 TABLETS 180 TABLETS

Month 2 180 TABLETS 180 TABLETS

Month 3 180 TABLETS 180 TABLETS

Month 4 180 TABLETS 180 TABLETS

Month 5 180 TABLETS 180 TABLETS

Month 6 180 TABLETS 180 TABLETS

TOTAL 1080 TABLETS 1080 TABLETS

Bottle of 90 Tablets 12 12

Assumed cost per bottle 26.00 318 318.00 cost per day (€) 1.77 1.77 3.53

*If due to untreated Lyme disease, then one has to understand that the Borrelia burgdorferi bacterium is not the only potential cause of the neurological disorder as the disorder is multi-factorial due to potential of co-infections caused by the initially weakened immune state of the individual. In Lyme disease, one has to diagnose the bacterium and then address the immune state of the patients with specific supplementation and diet change (16).

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1. Significant changes in cognitive state from day 0 to dayw 180. References 2. Significant changes in HSV-1 viral load. 1 American Chemical Society (2000, May 12). New Evidence Found Linking Herpes and Alzheimer’s. Science Daily. Retrieved July 26, 2013, 3. Significant changes in “well-being” i.e. rate of change in “senior from http://www.sciencedaily.com/releases/2000/05/000512083302.htm. moments experienced in a week”. 2 University of Manchester (2008, December 7). Cold Sore Virus Linked To D. Potential Daily Cost per day of Mushroom Alzheimer’s Disease: New Treatment, Or Even Vaccine Possible. Science Supplementation to Patient Daily. Retrieved July 26, 2013, from http://www.sciencedaily.com / releases/2008/12/081207134109.htm Supplementation at the price of a full treatment for six months would 3 MA Wozniak, AP Mee and RF Itzhaki. Herpes simplex virus type I DNA is be equal to two “Big Macs” and soft drinks per day. located within Alzheimer’s disease amyloid plaques. The Journal of Pathology, Volume 217, Issue 1 , Pages131 - 138 2009 DOI: 10.1002/path.2449 Concluding Remarks: 4 Carter CJ. Alzheimer´s disease plaques and tangles:cemeteries of phyrrhic In sum, given the fact that there may be a viral trigger for the onset victory of the immune defence network against herpes simplex infection at of Alzheimer´s disease and given the recently confirmed profile of the expense of complement and inflammation-mediated neuronal destruction. mushroom nutrition supplying both immune modulation and SOD Neurochem Int. 2011 Feb 58(3):301-20 doi 10 1016/j.neuint 2010.12.20.003 supplementation, it seems with worth the effort to Epub 2010 Dec 15. a) determine if mushroom nutrition does trigger lipoxin A4 activitiy in 5 Wozniak MA, Frost AL, Preston CM, Itzhaki RF. Antivirals reduce the formation the microglia; and if so: of key Alzheimer´s disease molecules in cell cultures acutely infected with b) conduct a clinical trial in early stage patients with an established herpes simplex virus type 1. PLoS One. 2011:6(10) e25152 doi 10 1371/ journal. (and recognized) parameters for success. Such parameters could be pone.0025152. EPUB 2011 Oct 7. a) decrease in onset of AD and b) decrease in rate of increase “tau” 6 Pandley JP. Immunoglobulin GM genese as functional risk and protective production. factors for the development of Alzheimer´s disease. J. Alzheimers Dis Conclusion 2009;17(4) 753-6 doi 10.3222/JAD-2009-1094. Given the expanding number of patients with Alzheimer´s disease 7 Wozniak MA, Itzhaki RF. Intravenous immounoglobulin reduces amyloid and and the increasing cost of care for Alzheimer patients, government abnormal tau formation caused by herpes simplex virus type 1. J Neuroimmunol healthcare services require safe, clinically reliable and cost effective 2013 Apr 15:257 (1-2):7-12 doi 10. 1016/ jnmeuroim 2013.01.005 Epub 2013 protocols that treat the symptoms of Alzheimer´s disease; the Feb 4. aforementioned two part protocol may offer a cost effective tool 8 Medeiros R, Kitazawa M, Passos GF, Baglietto-Vargas D, Cheng D, Cribbs OH, for the management of this condition in developing and developed LaFerla FM. Aspirin-triggered lipoxin A4 stimulates alternative activation of countries. microglia and reduces Alzheimer disease-like pathology in mice. Am J Pathol. 2013 May, 182(5);1740-9 .. 9 Jean A. Monro, Chronic Fatigue Immune Dysfunction Syndrome. J Integrative Medicine 2004; 8:101-108 10 Jean A. Monro. Treatment of Cancer with Mushroom Products. Arch Env Health 2003;58:533-537. 11 Silva Couto J, Pereira da Silva D. Evaluation of the Efficacy of Coriolus versicolor Supplementation in HPV Lesions (LSIL). Poster presented at the 20th European Congress of Obstetrics and Gynecology, Lisbon, Portugal March 4-8th, 2008. 12 Cornelius C. , Cavallaro M., Cambria M.T., Toscano M.A., Calabrese V. Comparative Enzyme Analysis of Polyporus umbellatus, Agaricus blazei, Pleurotus osteratus and Hericium erinaceus. Clinical Journal of Mycology 2009 Vol II Page 4 to 7. 13 Kulmann I, Minihane Am, Huebbe P, Nebel A , Rimbach G. Apolipoprotien E genotype and hepatitis c, HIV and herpes simplex diseae risk: a literature review. Lipids Health Dis. 2010 Jan 28.9.8 doi 10.1186/1476-511-9.6 14 Miklossy J. Emerging roles of pathogens in Alzheimer disease. Expert Rev Mol Med. 2011 Sept 20; 13 e 30 doi 10 1017/S14623994 1 1002006.

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